PROJECT SUMMARY The notion that not all obese individuals are at the same metabolic risk and that different types and/or degrees of weight loss alter this risk is debated. Genetic variation and metabolite profiles have been proposed to resolve metabolic risk in the obese, though the risk attributed to genetics and metabolites remains small. Novel markers that identify sub-phenotypes of metabolic risk in the obese are therefore necessary to better understand mechanism, determine the roll of weight loss, and uncover pathways for therapeutic targeting. Plasma extracellular RNAs (ex-RNAs) are circulating RNAs involved in trans-organ communication in obesity, epigenetically regulating a complex architecture of gene expression in adipocytes, hepatocytes, and skeletal muscle to reinforce metabolic syndrome. We recently used RNA sequencing and high-throughput RT-qPCR to identify plasma ex-RNAs associated with insulin resistance, visceral and hepatic fat, and obesity in several thousand individuals and validated the association with insulin resistance in an obese pediatric population and demonstrated alteration after extreme weight loss and diabetes resolution post bariatric surgery. In the U.S., the 2016 prevalence of obesity was 39.8% and affected about 93.3 million adults. Despite the benefits of surgical weight loss, its extensive risks preclude routine utilization and the vast majority of obese and overweight individuals employ behavioral and dietary approaches. Unknown is the impact of these significantly more common patterns of weight loss and potential regain on inflammatory/epigenetic markers including ex-RNAs that are associated with and contribute to metabolic risk. The Weight Loss Maintenance Randomized Controlled Trial was a two-phase study in which 1032 overweight or obese adults who had lost at least 4 kg during a 6-month weight loss program (phase 1) were randomized to a weight-loss maintenance intervention (phase 2). To determine the impact of behavioral weight loss and regain on circulating gene expression, we propose to; (1) determine if patterns of circulating ex-RNA expression from behavioral weight loss are similar to bariatric surgical weight loss; (2) determine if specific patterns of ex-RNAs predict individuals with sustained weight loss vs. regain; and (3) obtain preliminary proteomic data to confirm molecular targets. The central hypothesis of this proposal is that plasma ex-RNAs known to be associated with metabolic syndrome are altered after behavioral weight loss and detrimental patterns are influenced by changes in weight. This proposal will leverage strong existing data and utilize technologies and bioinformatics established in our laboratory to apply a transcriptomic approach to understand the molecular underpinnings of obesity and uncover potential novel mediators associated with non-surgical weight loss. Data from this project would be utilized to expand the study with validation in a broader population, contributing to the identification of potential molecular transcriptomic and proteomic targets for risk stratification and therapeutic exploration.